1.
Title :
Standard operating procedure for Calibration of HPLC
2. Purpose:
To lay down the procedure for the calibration of HPLC.
3. Scope:
This procedure shall be applicable for calibration of
HPLC.
4. Responsibility: Research Associate/Research Scientist.
5. Procedure :
5.1
Calibration of Pump.
5.1.1
Flow rate accuracy :
Calculate the flow
rate/min from each port ( A,B,C and D )by using water. Weigh the water collected by using 0.5 ml/min, 1.0
ml/min, 1.5 ml/min and 2.0 ml/min each for 5 minutes. The calibration is O.K.
if the values are within the limit.
5.1.2
Gradient accuracy test :
Preparation of Mobile phase
Mobile Phase A : Purified Water.
Mobile Phase B, C and D : 0.05% Acetone in purified water Carry out the
gradient composition test for Port
A&B, A&C and A&D and use the gradient program as mentioned in
the report
Format.
5.1
Calibration of Auto
injector :
5.1.1
Injector Linearity :
Mobile phase
preparation : Mix 850 ml of Acetonitrile with 150 ml of HPLC water. Filter through 0.45µL filter paper and degas.
Preparation of stock
solution :
Weigh accurately about
50 mg of caffeine standard in 100 ml volumetric flask. Add about 70 ml of
mobile phase. Sonicate to dissolve and diluted up to the mark with the same.
Preparation of 25 ppm
caffeine solution:
Pipette out 0.5 ml of
the above stock solution in 10 ml volumetric flask. Diluted up to the mark with
mobile phase.
Chromatographic conditions:
Column : Inertsil ODS-3, 250 X 4.6 mm X 5µ
Detector Wavelength : 273 nm
Flow Rate :
1.00 ml/min
Column Temperature : 40°C
Run
Time : 5
minutes
Inject the 25 ppm
caffeine solution 5.0 µL,10.0 µL, 20µL, 40µL 60 µL, 80µL and 100µ in
triplicate. Calculate co-relation coefficient Limit: NLT 0.999.
5.2.2 Injector
volume accuracy :
Chromatographic conditions : Same
as mentioned in 5.2.1.
Inject 50µl of water for six time with run time of 1.0 min. Calculate
the difference in the weight of the vial. Limit : +/- 1.0µL
5.2.3
Carryover test.
Chromatographic conditions:
As mentioned in 5.2.1
a)
Inject the blank solution.
b)
Prepare 500 ppm caffeine solution and inject 20µl of the solution.
c)
Inject the second blank.
d)
Calculated the % carryover. Limit NMT 0.01%
5.3
Column oven Temperature
Calibration
5.3.1. Set temperature at
20°C, 30°C, 40°C, 50°C and 60°C and observe the temperatures at the set temperatures. Limit is + /-
1°C.
5.4
Sample Compartment
Temperature Calibration :
5.4.1
Set the temperature of the sample compartment at 10°C and 20°C and note
down the temperature at the set temperatures. Limit is +/- 1°C.
5.5.
Calibration of Detector :
5.5.1
By using Caffeine
Solution ( 25 ppm )
a) Chromatographic conditions: As
mentioned in the 5.2.1.
b) Prepare 25 ppm caffeine solution
by using 0.5 ml of stock solution of 500 ppm and diluted to 10 ml volumetric
flask..
c) Inject 20µl of the above solution
at 270,271,272,273,274,275and 276 nm respectively in duplicates.
d) Maximum area should be observed at
273 nm +/- 1 nm.
5.5.2
Detector Linearity
a) Prepare concentration levels of
caffeine of 5, 25, 50,100 and 250 ppm. Inject the above solutions in
triplicates.
b) Chromatographic conditions as
mentioned in 5.2.1.
c) Limit : correlation coefficient
NLT 0.999.
6.0 Abbreviations:
1) HPLC: High Performance
Liquid Chromatograph
2) ppm
: Parts per million
7.0 Records:
|
Sr.No. |
Name of the Document |
Rev No. |
|
1 |
HPLC Calibration Report |
01 |
8.0 References
:
1) Shimadzu HPLC Manual.
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